Abstract

Background and Purpose Traumatic brain injury conveys significant morbidity and mortality to infants and children. In the newborn pig, opioids contribute to pial artery vasconstriction after fluid percussion injury (FPI). FPI attenuates vasodilation and cGMP production by methionine enkephalin (Met) and leucine enkephalin (Leu) and reverses dynorphin (Dyn) from a dilator to a constrictor. Superoxide anion (O2−) production contributes to altered cerebral hemodynamics after FPI, and O2− scavengers partially restore decreased dilator responses after FPI. Endothelin-1 (ET-1), a purported mediator of cerebral vasospasm, has been suggested to alter nitric oxide function and cGMP concentration. The present study was designed to determine the contribution of ET-1 to altered opioid-induced dilation after FPI and the role of O2− in such altered responses.

Methods Injury of moderate severity (1.9 to 2.3 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase (SOD)–inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O2− generation.

Conclusions These data show that ET-1, in concentrations similar to that present in cerebrospinal fluid after FPI, increases O2− production. These data also indicate the opioid-induced vasodilation and cGMP production are partially restored after FPI by ET-1 receptor blockade. These data suggest that ET-1 contributes to altered cerebral hemodynamics after FPI, at least in part, through elevated O2− production.

Traumatic brain injury accounts for significant morbidity and mortality in children younger than 1 year.1 Cerebral blood flow after traumatic head injury is thought to be a major contributor to outcome and is an important target in therapeutic strategies. For example, a decrease in cerebral blood flow has been described in children after brain injury.2 Cerebral concussion is an important sequela of brain injury in hospitalized patients, and the lateral brain FPI model mimics this entity with fidelity in animals.3 Previous studies have shown that FPI resulted in pial arterial vasoconstriction and decreased cerebral blood flow within 10 minutes of injury in newborn pigs.4 However, little is currently known about the mechanism of control of cerebral circulation in the newborn after traumatic brain injury.

Opioids appear to play an important role in control of the cerebral circulation in the newborn brain after FPI.5 Previous research has shown that during resting conditions, methionine enkephalin and leucine enkephalin are pial arterial vasodilators, while dynorphin is a tone-dependent agent (a dilator under resting tone, a constrictor when tone is decreased).6 Recently, it was observed that FPI alters the effects of opioids on the cerebral circulation. For example, the vasodilatory effects of methionine enkephalin and leucine enkephalin were blunted, while dynorphin became a vasoconstrictor after FPI.7 Opioid-induced pial artery vasodilation is mediated, in part, through the generation of cGMP by NO,8 the putative EDRF.9 O2− production is thought to antagonize NO function and to contribute to altered cerebral hemodynamics after FPI because O2− scavengers partially restored decreased dilator responses after FPI.10

ET, a 21–amino acid peptide with potent vasoconstrictor properties, was described in 1988 by Yanagisawa et al11 and subsequently found to comprise a family of pharmacologically distinct isoforms, ET-1, ET-2, and ET-3.12 Two receptors for ET, ETA and ETB, have been identified.13 The ETA receptor is highly specific for ET-1; stimulation of this receptor mediates most of the vasoconstrictor response to ET.14 The ETB receptor, unlike ETA, responds equally well to all of these isoforms of ET and mediates vasodilation by stimulating the release of NO and/or prostaglandins.1415 Previous work has demonstrated the prevalence of ET-1 in the central nervous system.1617 Although there are conflicting reports regarding whether ET levels are elevated as well as the efficacy of using ET antagonists, some evidence supports an etiologic role for ET-1 in vasospasm associated with subarachnoid hemorrhage.1819 Moreover, there is evidence for an increase in ET in the spinal cord after injury, while recent data link ET to blood–spinal cord barrier breakdown after traumatic injury.20 Such a local interaction is particularly important, since it is thought that the vasoactive properties of ET-1 are mediated along the abluminal front of blood vessels.21 Therefore, CSF ET concentration could serve as an index of its contribution to cerebral hemodynamics. Recently, it has been observed that FPI increased CSF ET-1 concentration and that BQ123, an ETA antagonist, attenuated pial artery constriction after FPI in the piglet.22 Although ET-1 has been suggested to alter NO function and cGMP concentration,2324 the mechanism by which this peptide might cause such alterations is uncertain.

Therefore, the present study was designed to determine the contribution of ET-1 to altered opioid-induced dilation after FPI and the role of O2− in such altered responses.

Materials and Methods

Fifty-four newborn pigs (1 to 5 days old) of either sex were used in these experiments. All protocols were approved by the Institutional Animal Care and Use Committee. Animals were anesthetized with ketamine hydrochloride (33 mg/kg) and acepromazine (3.3 mg) intramuscularly. Anesthesia was maintained with α-chloralose (30 to 50 mg/kg, supplemented with 5 mg/kg per hour IV). A catheter was inserted into a femoral artery to monitor blood pressure and to sample for blood gas tensions and pH. Drugs to maintain anesthesia were administered through a second catheter placed in a femoral vein. The trachea was cannulated, and the animals were mechanically ventilated with room air. A heating pad was used to maintain the animals at 37°C to 38°C.

One or two cranial windows were placed in the parietal skull of these anesthetized animals. The window consists of three parts, a stainless steel ring, a circular glass coverslip, and three ports consisting of 17-gauge hypodermic needles attached to three precut holes in the stainless steel ring. For placement, the dura was cut and retracted over the cut bone edge. The cranial window was placed in the opening and cemented in place with dental acrylic. The space under the window was filled with artificial CSF of the following composition (in mg/L): KCl 220, MgCl2 132, CaCl2 221, NaCl 7710, urea 402, dextrose 665, and NaHCO3 2066. This artificial CSF had the following chemistry: pH 7.33, Pco2 46 mm Hg, and Po2 43 mm Hg, which was similar to endogenous CSF. Pial arterial vessels were observed with a dissecting microscope, a television camera mounted on the microscope, and a video monitor. Vascular diameter was measured with a video microscaler (model VPA 550, For-A-Corp).

Methods for brain FPI have been described previously.25 A device designed by the Medical College of Virginia was used. A small opening in the parietal skull contralateral to the cranial window(s) was made. A metal shaft was sealed into the opening on top of intact dura. This shaft was connected to transducer housing, which was in turn connected to the fluid percussion device. The device itself consisted of an acrylic plastic cylindrical reservoir 60 cm long, 4.5 cm in diameter, and 0.5 cm thick. One end of the device was connected to the transducer housing, while the other end had an acrylic plastic piston mounted on O-rings. The exposed end of the piston was covered with a rubber pad. The entire system was filled with 0.9% saline (37°C). The percussion device was supported by two brackets mounted on a metal platform. FPI was induced by striking the piston with a 4.8-kg pendulum. The intensity of the blow (usually 1.9 to 2.3 atm with a constant duration of 19 to 23 milliseconds) was controlled by varying the height from which the pendulum was allowed to fall. The pressure pulse of the blow was recorded on a storage oscilloscope triggered photoelectrically by the fall of the pendulum. The amplitude of the pressure pulse was used to determine the intensity of the injury.

Protocol

Drug effects on the diameter of two types of pial arterial vessels—small arteries (baseline diameter, 120 to 160 μm) and arterioles (baseline diameter, 50 to 70 μm)—were examined to determine whether segmental differences in the actions of ET-1 could be identified. Pial arterial vessel diameter was determined every minute for a 10-minute exposure period after infusion onto the exposed parietal cortex of artificial CSF containing no drug and after infusion of artificial CSF containing a drug. A volume of 1 to 2 mL of artificial CSF was then flushed over 30 seconds through a port in the cranial window, and approximately 300 μL from another port at the end of the 10-minute period was collected. The collected samples were analyzed for cGMP concentration.

We performed five major types of experiments: (1) FPI without BQ123 pretreatment (n=7); (2) FPI with BQ123 pretreatment (n=7); (3) time control (n=5); (4) generation of O2− with FPI (n=15); and (5) generation of O2− with ET-1 (n=10).

Responses of arterial vessels to methionine enkephalin, leucine enkephalin, and dynorphin at concentrations of 10−10, 10−8, and 10−6 mol/L (Sigma Chemical Co) were obtained before and 1 hour after FPI with and without pretreatment with 10−6 mol/L BQ123 solution (RBI). In animals that were treated with BQ123, this antagonist was administered 20 minutes before FPI. Each of the opioids was applied in an ascending concentration manner. There was a period of 20 minutes after the highest concentration of one opioid was washed off before a different opioid was infused. The percent changes in artery diameter values were calculated based on the diameter measured in the control period for each drug before injury for preinjury (control) values; the diameter present in the control period before drug administration after injury was used for brain injury values. Time-control experiments were conducted in a separate series of animals and were designed to obtain responses to drugs initially and then 1 hour later. Finally, in a separate series of animals, the production of O2− after FPI with and without pretreatment with BQ123 was examined. Additionally, the ability of ET-1 (Sigma) to generate O2− was investigated. Normal (0.9%) saline was used as a vehicle for all drugs, and it had previously been observed to have no effect on arterial vessel diameter.7 All working drugs were made fresh on the day of use.

cGMP Analysis

Artificial CSF samples collected after a 10-minute exposure to an opioid were analyzed for cGMP according to scintillation proximity assay methods. Commercially available kits for cGMP (Amersham) were used. Briefly, this assay determines cyclic nucleotide concentration for binding to an antiserum that has a high specificity for cGMP. The antibody-bound cyclic nucleotide was then reacted with a second anti-rabbit antibody bound to fluoromicrospheres. Labeled cyclic nucleotide bound to primary rabbit antibody can be measured by determining the amount of light emitted by the fluoromicrospheres. All unknowns were assayed at two dilutions. The concentrations of unlabeled cyclic nucleotides were calculated from the standard curve by linear regression analysis.

O2− Analysis

SOD-inhibitable NBT reduction was determined as an index of O2− generation as previously described.1026 In one group of animals, O2− generation was determined in the first 20 minutes after application of 10−10 mol/L ET-1. In addition, O2− generation in the first 20 minutes after a 1-hour waiting period after FPI in animals treated with vehicle (saline) and in piglets that were treated with BQ123 (10−6 mol/L) 20 minutes before FPI was determined. In a fourth group of pigs, SOD-inhibitable NBT reduction was determined for 20 minutes without prior FPI (control). SOD-inhibitable NBT reduction was determined by placing NBT (Sigma, 2.4 mmol/L) dissolved in artificial CSF under one window and NBT (2.4 mmol/L) and SOD (Sigma, 60 U/mL) in artificial CSF under the other window 1 hour after FPI. Two windows were placed contralateral to the adapter for induction of FPI for these experiments.

NBT is water soluble and forms a yellow solution that is converted to nitroblue formazan, an insoluble purple precipitate, in the presence of reducing agents, eg, O2−. The SOD-inhibitable NBT reduction was determined by the difference in the quantities of nitroblue formazan precipitated on the brain surface under the two windows. Although NBT can be reduced by a variety of agents, SOD provides specificity for the assay. Slices of the brain surface 1 mm thick under each cranial window were obtained. The slices were minced and homogenized in 1N NaOH and 0.1% sodium dodecyl sulfate solution. The homogenate was centrifuged at 20 000g for 20 minutes. The supernatant was discarded, and the pellet was resuspended in 3 mL of pyridine. The formazan was dissolved in the pyridine during heating at 80°C for 1 hour. Particulate matter was removed by a second centrifugation at 10 000g for 10 minutes. The concentration of nitroblue formazan in the supernatant was then determined spectrophotometrically at 515 nm. The nitroblue formazan on the NBT alone side was analyzed against the background of the SOD-treated side. Freshly prepared calibration solutions were used with each set of samples and treated identically to the samples. Recovery of NBT averaged 88±4%.

Statistical Analysis

Pial artery diameter, systemic arterial pressure, cyclic nucleotide levels, and amount of NBT reduced were analyzed with ANOVA for repeated measures. If the value was significant, Fisher's exact test was performed. A value of P<.05 was considered significant. The n values reflect data for one vessel in each animal. Values are represented as mean±SEM of absolute values or as percentages of change from control values. Data presented as percent change were compared by nonparametric means with the Wilcoxon signed rank test.

Results

O2− Generation After ET-1 and FPI

SOD-inhibitable NBT reduction was increased after topical administration of ET-1 (10−10 mol/L), a concentration similar to that previously observed in cortical periarachnoid CSF 60 minutes after FPI22 (Fig 1A⇓). SOD-inhibitable NBT reduction was also increased after FPI, and this increase was blunted by pretreatment with BQ123 (10−6 mol/L) (Fig 1B⇓).

A, Determination of SOD-inhibitable NBT reduction in newborn piglet brain before (control) and after topical ET-1 (10−10 mol/L) (n=5). B, Determination of SOD-inhibitable NBT reduction in piglet brain before (control) and after FPI and after FPI in BQ123-pretreated animals (FPI-BQ123) (n=5). *P<.05 compared with control; +P<.05 compared with untreated animals.

Influence of BQ123 on Opioid-Induced Pial Artery Dilation After FPI

Methionine enkephalin (10−10, 10−8, and 10−6 mol/L) elicited reproducible pial small-artery (120 to 160 μm) and arteriole (50 to 70 μm) vasodilation (Table⇓). These increases in vessel diameter were attenuated after FPI and partially restored by BQ123 (Fig 2⇓). Methionine enkephalin produced dilation that was associated with increased cortical periarachnoid CSF cGMP, and these biochemical changes were blunted by FPI and partially restored by BQ123 (Fig 3⇓). Leucine enkephalin (10−10, 10−8, and 10−6 mol/L) also produced pial artery dilation (Table⇓) that was similarly attenuated after FPI and partially restored by BQ123 (Fig 4⇓). Leucine enkephalin–induced dilation was associated with an increase in CSF cGMP, which was attenuated after FPI and partially restored by BQ123 (Fig 5⇓). These values represent a change in CSF cGMP similar to that produced with methionine enkephalin.

Influence of methionine enkephalin (10−10, 10−8, and 10−6 mol/L) on small pial arteries and arterioles before (control), after brain FPI, and after FPI in BQ123-pretreated animals (FPI-BQ123) (n=7). *P<.05 compared with corresponding control value; +P<.05 compared with corresponding FPI value.

Influence of leucine enkephalin (10−10, 10−8, and 10−6 mol/L) on small pial arteries and arterioles before (control), after brain FPI, and after FPI in BQ123-pretreated animals (FPI-BQ123) (n=7). *P<.05 compared with corresponding control value; +P<.05 compared with corresponding FPI value.

In contrast, dynorphin (10−10, 10−8, and 10−6 mol/L), which is a vasodilator under resting conditions (Table⇑), decreased pial artery diameter after FPI, and this opioid was restored to a vasodilator by BQ123 (Fig 6⇓). Dynorphin produced dilation that was associated with a large increase in CSF cGMP. This biochemical change was blunted after FPI and partially restored by BQ123 (Fig 7⇓).

Influence of dynorphin (10−10, 10−8, and 10−6 mol/L) on small pial arteries and arterioles before (control), after brain FPI, and after FPI in BQ123-pretreated animals (FPI-BQ123) (n=7). *P<.05 compared with corresponding control value; +P<.05 compared with corresponding FPI value.

Influence of dynorphin (10−10, 10−8, and 10−6 mol/L) on cGMP concentration in cortical periarachnoid CSF before (control), after brain FPI, and after FPI in BQ123-pretreated animals (FPI-BQ123) (n=7). *P<.05 compared with corresponding control value; +P<.05 compared with corresponding FPI value.

Influence of BQ123 on Pial Artery Diameter and CSF cGMP After FPI

In five piglets, FPI decreased pial small-artery diameter from 145±7 to 122±5 and pial arterioles from 61±2 to 52±1 μm 1 hour after injury. In contrast, FPI only decreased pial small-artery diameter from 136±7 to 129±3 and arteriole diameter from 64±2 to 59±3 μm in BQ123-pretreated animals. Similarly, FPI decreased resting control values for cGMP, and BQ123 pretreatment partially prevented those decreases (Figs 3, 5, and 7⇑⇑⇑).

Influence of BQ123 on the Pial Artery Response to ET-1

In five piglets, ET-1 elicited 8±1% and 11±1% decreases in pial small-artery and arteriole diameter, respectively. These decreases in diameter were blocked by BQ123 (1±1% and 1±1% decrease for small arteries and arterioles, respectively).

Blood Chemistry

Blood chemistry values were obtained at the beginning and end of all experiments. These values were 7.42±0.01, 30±2, and 98±2 versus 7.43±0.02, 31±1, and 98±3 versus 7.41±0.01, 32±1, and 92±2 for pH, Pco2, and Po2 before FPI, after FPI, and at the end of the experiment, respectively (n=37). Mean arterial blood pressure decreased from 68±1 to 55±1 mm Hg within 60 minutes of FPI (n=37). The mean level of FPI was 1.9±0.1 atm.

Discussion

Results of the present study show that ET-1 increased SOD-inhibitable NBT reduction by newborn pig brains, indicating that O2− radical was generated. The concentration of ET-1 chosen for the above experiments was one that approximated the concentration of ET-1 observed in cortical periarachnoid CSF 1 hour after brain injury.22 In turn, this concentration of ET-1, 10−10 mol/L, increased SOD-inhibitable NBT reduction to an extent similar to that observed 1 hour after injury.10 Moreover, the ETA receptor antagonist BQ123 blunted brain injury–induced elevated SOD-inhibitable NBT reduction. BQ123 had previously been shown to selectively inhibit ETA receptor–mediated pial artery vasoconstriction by ET-1,22 an observation confirmed in the present study. Taken together, these data suggest that ET-1 released into CSF after brain injury is an important contributor to the generation of O2− after injury.

Results of this study also show that methionine enkephalin and leucine enkephalin elicited pial artery dilation associated with increased CSF cGMP and that these vascular and biochemical changes were attenuated by brain injury, consistent with previous observations.710 In contrast, pretreatment with BQ123 partially restored the opioid-induced dilation and ability to increase CSF cGMP after brain injury. Moreover, dynorphin was reversed from a dilator to a constrictor after brain injury, and this response was also partially restored by BQ123. Previously, it has been observed that altered opioid-induced vasodilation and cGMP production were partially restored with the preadministration of the oxygen radical scavengers polyethylene glycol SOD and catalase,10 indicating a role for such radicals in altered opioid dilation after injury. Moreover, these radical scavengers also attenuated the reductions in baseline CSF cGMP and pial vessel diameter observed after brain injury,10 indicating that the generation of oxygen radicals contributes to the reduction in tonically released NO and pial artery constriction after injury. Results of the present study show that BQ123 similarly attenuated such brain injury–associated reductions in CSF cGMP and pial artery diameter. These data, therefore, suggest that ET-1 production after brain injury alters opioid-induced pial artery dilation by superoxide generation.

The cerebrovascular consequences of free radical production are not fully understood. However, there is a significant amount of evidence that supports a role of oxygen radicals in brain injury. For example, brain injury in cats has been reported to cause the generation of superoxide for at least 1 hour after injury.27 In that study, the sustained dilation and abnormal responsiveness of pial arterioles observed after injury could be reversed by treatment with the free radical scavengers SOD and catalase.27 Oxygen radicals also have been shown to increase blood-brain barrier permeability,28 produce ultrastructural changes in pial vessel endothelium,29 and cause abnormal arteriolar reactivity.29 In addition, oxygen radical scavengers have been shown to improve vascular function and blood flow during focal ischemia in rats, which may account for the observed reductions in infarct size.30 Recently, a trial with SOD in humans with severe head injuries showed that death and vegetative state were increased in patients receiving a placebo compared with those receiving polyethylene glycol and SOD.31

The mechanism for the altered cerebral hemodynamics observed after brain injury has been investigated previously. For example, functional alterations have been accompanied by abnormalities in endothelial morphology and impairment of endothelium-dependent relaxation,2532 suggesting that altered release of EDRF contributes to the reduction in cerebral blood flow after brain injury. Recently, it has been observed that NO, an example of an EDRF, contributes to opioid-induced pial artery dilation.8 In the newborn pig, opioids appear to play an important role in the control of the cerebral circulation. For instance, CSF concentrations of opioids are in the vasoactive range under control conditions, and opioids contribute to the vascular actions of physiological stimuli such as hypoxia and hemorrhagic hypotension.633 Intracellular generation of O2− or other species could alter structures and/or production of nucleotides, second messengers, receptors, and membranes, and the movement of O2− out of the cell through anion channels could result in high concentrations of activated oxygen species at cell surfaces, including endothelium. Such oxygen species, then, may alter opioid-related NO generation metabolism or action.

Previous studies have characterized the hemodynamic effects of brain injury in adult animals.253234 However, few studies have investigated the effects of brain injury in the newborn/infant time period. The present study may approximate the human newborn/infant time period because both newborn pigs and children younger than 1 year have skulls with unfused sutures. It has been observed that developmental changes result in markedly different effects of brain injury on cerebral hemodynamics in the newborn and juvenile pig.4 For example, it was observed that pial vessels constricted more and regional blood flow decreased and remained depressed longer in newborns than in juveniles. Furthermore, systemic arterial pressure has been observed to increase in adult studies2528 and in juvenile pigs,4 whereas results of the present study show that systematic arterial pressure decreases after brain injury in the newborn pig, consistent with previous newborn studies.451022 Therefore, cerebral and systematic hemodynamic responses after brain injury are age dependent.

The role of the systemic pressor response in altered adult cerebral hemodynamics has been investigated. For example, it was hypothesized that acute elevations of blood pressure after injury in the adult result in the release and metabolism of arachidonic acid, which would generate oxygen free radicals, causing cerebral functional abnormalities.25272835 Results of the present study confirm that superoxide is generated 1 hour after brain injury in the newborn pig. However, in contrast to studies performed in adult and juvenile animals, there was no acute elevation in blood pressure after injury, which corroborates previous studies in the newborn pig.451022 In contrast, brain injury results in a decrease in mean arterial pressure, which remains depressed for up to 3 hours after injury.45

Since the elevation in systematic blood pressure was thought to be an absolute requirement for cerebral generation of oxygen radicals after injury,25283235 the observed decrease in blood pressure associated with radical release in the newborn pig was perplexing. Results of the present study, therefore, are the first to link the cerebral release of a peptide, ET-1, to superoxide generation and thus serve as a partial explanation for the above perplexity.

Recent studies have indicated an antagonistic relationship between ET and endothelium-dependent mechanisms, including NO. For example, ET-1 reduced the ability of sodium nitroprusside, an NO releaser, to elevate cGMP concentration in human pulmonary vessels, suggesting that this peptide may play a role in the control of pulmonary vascular tone by modifying cGMP levels associated with vasorelaxant agonist stimulation.24 Additionally, ET-1 was produced after ischemia and reperfusion in isolated rabbit hearts, while BQ123 preserved the decreased responsiveness of coronary resistance vessels to the endothelium-dependent dilator acetylcholine after ischemia and reperfusion.23 In that study, it was suggested that ET-1 impaired formation or enhanced the degradation of EDRF in the setting of ischemia and reperfusion.23 Moreover, in a rabbit model of subarachnoid hemorrhage, it was observed that BQ123 partially reversed the associated vasospasm and restored the magnitude of acetylcholine relaxation of basilar arteries.36 There are several possible mechanisms by which this might occur. First, endogenous ET-1 could enhance the production of oxygen free radicals, particularly superoxide. It has previously been observed that superoxide radical can degrade EDRF.37 While the method by which ET-1 could generate superoxide is uncertain, it appears to be selective for this peptide. For example, it has been reported that injury to ischemic/reperfused rat skeletal muscle was worsened by both ET-1 and angiotensin II infusion. However, only ET-1–mediated injury was reduced by oxygen radical scavengers.38 A second possible explanation for the observed interaction between NO and ET-1 involves a reduction in NO release by ET-1. A third and final possible mechanism involves unopposed stimulation of the ETB receptor by endogenous ET-1 during ETA receptor blockade with BQ123. This would be expected to result in enhanced basal and/or stimulated NO release and could account for the observed beneficial effect of BQ123. While the latter two mechanisms remain a possibility, data from the present study support the first explanation for the observed findings.

Opioids themselves also have been investigated for their contributory role in the cerebral hemodynamic effects of brain injury. For example, the opioid antagonist naloxone has been observed to improve blood chemistry, electroencephalographic parameters, and brain perfusion pressure in cats39 ; attenuate pial artery constriction and reductions in cerebral blood flow after brain injury in piglets5 ; and improve long-term neurobehavioral outcome after brain injury in rats.40 Regional increases in dynorphin immunoreactivity in the parietal and frontal cortex, pons, medulla, and striatum were found to correlate with local histopathologic damage and reductions in cerebral blood flow after brain injury in the adult cat,41 suggesting that dynorphin and the κ-opioid system could play a role in the injury process after brain trauma. Further evidence in support of this concept is found in the observations that administration of dynorphin or the synthetic κ-agonist U50,488 H worsens neurological outcome,42 whereas the κ-antagonist nalmafene improves neurological outcome and metabolism after brain injury.43 Alternatively, U50,488 H also has been reported to improve spinal cord blood flow and neurological recovery after brain injury in mice.44 A partial explanation for these contradictory data could be that different κ-isoreceptors mediate different physiological effects and the above drugs could have varying affinities for these receptors.42 Recent studies in the newborn pig show that CSF opioid concentrations increase after brain injury and that the time course and relative increase in CSF concentration vary from opioid to opioid.5 Data from a recent study7 suggest that methionine enkephalin and leucine enkephalin produce pial dilation that would be beneficial, serving as a physiological antagonist to brain injury and pial artery constriction. Although the CSF concentration of these two opioids is increased after brain injury, their beneficial role is decreased because dilation by these opioids is attenuated after brain injury.7 Moreover, brain injury reversed dynorphin from a dilator to a constrictor, further contributing to pial artery vasoconstriction after injury.

In conclusion, results of the present study show that ET-1, in concentrations similar to those present in CSF after brain injury, increases superoxide production. These data also indicate that opioid-induced vasodilation and cGMP production are partially restored after brain injury by ET-1 receptor blockade. These data suggest that ET-1 contributes to altered cerebral hemodynamics after injury, at least in part, through elevated superoxide production.

Selected Abbreviations and Acronyms

CSF

=

cerebrospinal fluid

EDRF

=

endothelium-derived relaxing factor

ET

=

endothelin

FPI

=

fluid percussion injury

NBT

=

nitroblue tetrazolium

NO

=

nitric oxide

O2−

=

superoxide anion

SOD

=

superoxide dismutase

Acknowledgments

This study was supported by grants from the National Institutes of Health and the American Heart Association. Dr Armstead is an Established Investigator of the American Heart Association. The authors thank Joseph Quinn for technical assistance in the performance of the experiments.

Editorial Comment

Given the prominent role of ET-1 as a vasoconstrictor in vitro1R2R3R and in vivo,4R5R6R7R8R9R10R11R12R it is not surprising that there has been considerable interest in the role of this peptide in cerebral ischemia (see reviews in References 13-16). Elevated levels of ET have been reported both in focal ischemic brain tissue and in the extracellular fluid in global ischemia17R and focal cerebral ischemia.12R18R Application of ET-1 to the abluminal surface (tissue side) of cerebral vessels results in a dose-dependent, sustained reduction in blood flow9R10R12R19R20R concomitant with indications of ischemic neural damage.7R10R11R19R20R

More recently, ET has been implicated in secondary pathogenesis after traumatic injury to the central nervous system. An increase in ET has been reported in the CSF compartment after traumatic brain injury21R and in the parenchyma of the traumatized spinal cord.22R Although the role of ET in these traumatic states has yet to be clearly elucidated, there is growing evidence that the peptide may modulate blood-brain barrier function22R and mediate vasoconstriction.21R The present study takes an important step in defining the contribution of ET-1 to altered cerebral blood flow after traumatic brain injury in the neonatal pig. Although decreased cerebral blood flow has been reported in children after brain injury, little is known about the events that mediate the altered hemodynamics. It has been previously established that opioids are integral to the control of cerebral circulation in the neonatal brain after experimental brain FPI.21R The dilatory effects of methionine enkephalin and leucine enkephalin are blunted after trauma, and dynorphin, a tone-dependent agent, behaves as a vasoconstrictor. The present study is particularly interesting because it provides a putative link between elevated levels of ET-1 after traumatic brain injury and vascular responsiveness to opioids. In particular, it is shown that BQ123, an ETA receptor antagonist, significantly restores the normal vascular responsiveness to these opioids. Further evidence is provided which suggests that ET-1 modulates this vascular responsiveness through the generation of superoxides.